Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin

Our plain-English summary of Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin, by A. Shukla, J.M. Hillegass, M.B. MacPherson, et al (Molecular Cancer, 2010).

The study

• Authors: A. Shukla, J.M. Hillegass, M.B. MacPherson, S.L. Beuschel, P.M. Vacek, H.I. Pass, M. Carbone, J.R. Testa, B.T. Mossman
• Journal: Molecular Cancer
• Published: 2010;9:314
• Cited by: 65 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Malignant mesotheliomas (MM) have a poor prognosis, largely because of their chemoresistance to anti-cancer drugs such as doxorubicin (Dox). Here we show using human MM lines that Dox activates extracellular signal-regulated kinases (ERK1 and 2), causally linked to increased expression of ABC transporter genes, decreased accumulation of Dox, and enhanced MM growth. Using the MEK1/2 inhibitor, U0126 and stably transfected shERK1 and shERK2 MM cell lines, we show that inhibition of both ERK1 and 2 sensitizes MM cells to Dox.

Results: U0126 significantly modulated endogenous expression of several important drug resistance (BCL2, ABCB1, ABCC3), prosurvival (BCL2), DNA repair (BRCA1, BRCA2), hormone receptor (AR, ESR2, PPARγ) and drug metabolism (CYP3A4) genes newly identified in MM cells. In comparison to shControl lines, MM cell lines stably transfected with shERK1 or shERK2 exhibited significant increases in intracellular accumulation of Dox and decreases in cell viability. Affymetrix microarray analysis on stable shERK1 and shERK2 MM lines showed more than 2-fold inhibition (p ≤ 0.05) of expression of ATP binding cassette genes (ABCG1, ABCA5, ABCA2, MDR/TAP, ABCA1, ABCA8, ABCC2) in comparison to shControl lines. Moreover, injection of human MM lines into SCID mice showed that stable shERK1 or shERK2 lines had significantly slower tumor growth rates in comparison to shControl lines after Dox treatment.

Conclusions: These studies suggest that blocking ERK1 and 2, which play critical roles in multi-drug resistance and survival, may be beneficial in combination with chemotherapeutic drugs in the treatment of MMs and other tumors.

In plain English

This laboratory study used human malignant mesothelioma (MM) cell lines and mice with tumors grown from them. It did not study asbestos exposure. Doxorubicin (Dox), a chemotherapy drug, activated the signaling proteins ERK1 and ERK2 in four MM cell lines. In three lines, Dox plus U0126, a synthetic compound blocking this pathway, killed significantly more cells than either alone. HMESO and PPMMill cells engineered to make less ERK1 or ERK2 survived Dox worse than controls. After Dox, ERK2-reduced lines showed significantly greater killing than ERK1-reduced lines. ERK-reduced HMESO cells also kept more Dox inside. Without Dox, the two ERK-reduced lines together showed lower messenger RNA levels of seven ABC transporter genes than controls; the authors suggested each ERK regulated distinct genes. In SCID mice (3 mice, 12 tumors per group), Dox injected at the tumor sites slowed tumor growth in all groups, most in controls. Even so, Dox-treated tumors from ERK-reduced cells grew significantly more slowly than Dox-treated control tumors. The authors concluded that ERK inhibitors plus chemotherapy might treat MM better than drugs alone.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study using human mesothelioma cell lines in culture (in vitro) and a SCID mouse xenograft model (in vivo)
• Who was studied: Four human malignant mesothelioma cell lines: MO (sarcomatoid, pleural), ME-26 (epithelioid, pleural), HMESO (epithelioid) and PPMMill (sarcomatoid). The comparison cells were LP9/TERT-1, a line resembling normal human mesothelial cells. In the mouse experiment, HMESO cells were injected into SCID mice, N = 6 per group, split into 3 Dox-treated and 3 saline-treated mice, giving 12 tumors per group.
• Exposure: Not an asbestos exposure study. The exposures were the chemotherapy drug doxorubicin and blocking of ERK1/ERK2. Doxorubicin was given at 0 to 100 μM to cells, and at 6 μg per tumor site 3 times weekly, injected locally at the tumor sites, in mice. ERK1/ERK2 were blocked either by the synthetic MEK1/2 inhibitor U0126 (20 μM) added to cells or by stable shERK1 and shERK2 gene silencing.
• Main results:
  • The approximate dose of Dox that killed half the cells (LD50) was 25 μM for the MO and ME-26 lines, and approximately 100 μM or greater for the HMESO and PPMMill lines.
  • Dox activated (phosphorylated) ERK1 and ERK2. In the MO line this was significant (p less than or equal to 0.05) from the lowest Dox concentration used (1 μM).
  • In MO, ME-26 and HMESO cells, the synthetic MEK1/2 inhibitor U0126 plus Dox caused significantly more cell killing than Dox or U0126 alone.
  • In HMESO and PPMMill cells treated with Dox, reducing ERK1 or ERK2 significantly increased Dox-induced cell killing. The shERK2 lines showed significantly greater killing than the shERK1 lines.
  • HMESO cells with reduced ERK1 or ERK2 accumulated more Dox inside the cells than control cells.
  • In microarray analysis of HMESO cells not exposed to Dox, seven ABC transporter genes had decreased mRNA levels across the shERK1 and shERK2 lines compared with control lines. The authors suggested that different ERKs regulate distinct ABC genes.
  • Compared with LP9/TERT-1 mesothelial cells, the MO and ME-26 lines had significantly greater levels of many prosurvival and drug resistance genes. U0126 significantly altered levels of some of these genes.
  • In mice given localized Dox injections at the tumor sites, Dox significantly reduced the tumor growth rate in all three groups compared with saline, with the largest reduction in the shControl group.
  • Among Dox-treated mice, tumors from the shERK1 or shERK2 groups grew significantly more slowly than tumors from the shControl group.
• The authors' conclusion: The authors concluded that ERK1 and ERK2 play significant roles in Dox resistance of MM cells. They also concluded that ERK1/2 inhibitors combined with chemotherapy drugs might be a better option for treating MM patients than drugs alone.
• Limitations the authors state:
  • The authors stated that a detailed study is needed to understand how different ERKs regulate ABC transporter genes.
  • The authors noted that Dox retention was similar in shERK1 and shERK2 cells. They suggested that the greater Dox toxicity in shERK2 cells could be due to additional factors.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, Doxorubicin, Prognosis.

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• Microspheres targeted with a mesothelin antibody and loaded with doxorubicin reduce tumor volume of human mesotheliomas in xenografts — S.L. Macura, J.L. Steinbacher, et al. (2013)
• Decrease in phosphorylation of ERK following decreased expression of NK cell-activating receptors in human NK cell line exposed to asbestos — Y. Nishimura, M. Maeda, et al. (2009)
• Extracellular signal-regulated kinase 5 and cyclic AMP response element binding protein are novel pathways inhibited by vandetanib (ZD6474) and doxorubicin in mesotheliomas — M. Sayan, A. Shukla, et al. (2014)
• 3-O-Methylfunicone, a metabolite produced by Penicillium pinophilum, modulates ERK1/2 activity, affecting cell motility of human mesothelioma cells — E. Buommino, I. Paoletti, et al. (2010)
• An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury — A. Shukla, T.F. Barrett, et al. (2011)

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